RNA-seq of coding RNA of Streptococcus pneumoniae D39 after FCS addition
RNA-seq of coding RNA of Streptococcus pneumoniae D39 after FCS addition
Bacteria were harvested in ice-cold killing buffer (50 mM Tris pH 7.5, 5 mM MgCl2, 20 mM NaN3), centrifuged at 4750 rpm for 10 min at 4°C and the pellets were immediately frozen in liquid nitrogen and stored at −80°C.  S.p. were cultivated in supplemented RPMI medium in the presence or absence of 10% FCS, which was added to the +FCS culture at an OD600 of 0.3. The control1 culture was harvested prior to FCS treatment. The +FCS and –FCS cultures were harvested after 2 hours of growth after reaching OD600 of 0.3.  The bacteria were not treteated at an OD600 of 0.3 with FCS RNA isolation was performed using an acidic phenol-chloroform extraction protocol (Wetzstein et al., 1992). After DNase-I treatment (Zymo Research, Germany), the RNA quality was checked by Trinean Xpose (Gentbrugge, Belgium) and the Agilent RNA Nano 6000 kit using an Agilent 2100 Bioanalyzer (Agilent Technologies, Böblingen, Germany).  For RNA-seq transcriptomics, Ribo-Zero rRNA Removal Kit (Bacteria) from Illumina (San Diego, CA, USA) was used to remove the rRNA. TruSeq Stranded mRNA Library Prep Kit from Illumina (San Diego, CA, United States) was applied to prepare the cDNA libraries.